Electrophoresis system and method



Feb.24,1 970 J in. 3,497,441

ELECTROPHORESIS SYSTEM AND METHOD Filed May 16, 1967 2 sheets-sheet 1Fig. 4

INVENTOR.

JOSE/9H PAKS/ BY WM, Q/Og/MMW 3W M460 ATT QMEYs.

J. PAKSI ELEOTROPHORESIS SYSTEM AND METHOD Feb. 24, 1970 2 Sheets-Sheet2 Filed May 16, 1967 INVENTOR. v JOSE/ H PAKSI BY v a ATTORNEYS.

m a /wq United States Patent 7" 3,497,441 ELECTROPHORESIS SYSTEM ANDMETHOD Joseph Palrsi, 2217 Westminster Road, Cleveland Heights, Ohio44118 Filed May 16, 1967, Ser. No. 638,815 Int. 'Cl. B01k /00 US. Cl.20418fl 9 Claims ABSTRACT OF THE DISCLOSURE An electrophoresis systemfor separation analysis of solutions of ionized particles. A starch-gelmedium is formed on a Mylar substrate and separated by channels intotest strips. Under an electric current, particles of a sample materialmigrate different distances through the medium. The strips can bescanned in photoelectric analyzers. A drum applicator and cutting boardfacilitate preparation.

BACKGROUND OF THE INVENTION Field of the invention This inventionrelates to electrophoresis, which is the migration of particles underthe influence of an electric field. More particularly, this inventionrelates to methods and apparatus for utilizing electrophoresis toanalyze solutions of ionized particles, especially biochemicalmaterials.

Description of the prior art Electrophoresis is a known and importantanalytical technique. It has found particular usefulness in the medicalfield because it provides a means of performing the separation ofproteins, amino acids, nucleic acids, peptides, and other importantbiochemical materials. Four known types of electrophoresis are: (1)moving boundary electrophoresis, (2) zone electrophoresis, (3) twodimensional electrophoresis, and (4) immunoelectrophoresis.

Moving boundary electrophoresis permits quantitative estimation of themobilities and concentrations of various components, as in a mixedprotein solution, such as plasma. Physical channels are provided for thesolution and .movement of the particles in the solution is followed whenan electric current is past through the material.

Zone electrophoresis uses a supporting medium in which ions of thesolution migrate and separate into zones, depending upon their mobilityin an electric field. As compared with moving boundary techniques, theamount of material to be analyzed is very small. Turbid or highlypigmented samples cannot be satisfactorily illuminated for movingboundary electrophoresis. No such problem arises with zoneelectrophoresis techniques.

Two dimension electrophoresis combines two methods of zoneelectrophoresis into a single method to provide a more completeresolution of the components of the material being tested. Basically,components are separated in a first direction, then transferred to asecond medium, where the electrophoresis is continued at a right angleto the previous one for additional time.

Immunoelectrophoresis is used to study the complexity of antigens orantibodies through the use of zone electrophoretic techniques.

The present invention relates to zone electrophoresis, and many types ofzone electrophoresis are already known. These known types of zoneelectrophoresis include 1) paper electrophoresis, (2) cellulose acetate3,497,441 Patented Feb. 24, 1970 membrane electrophoresis, (3) gelelectrophoresis, including agar-gel, polyacrylamide gel and starch-gelblock electrophoresis, and (4) preparative electrophoresis, includingstarch block, sponge rubber, and continuous flow electrophoresis. Thedifferent types of zone electrophoresis techniques indicate the media inwhich the material to be analyzed migrates. These different techniquesvary in their sensitivity, convenience, and speed. For example, innormal human serum, five protein fractions can be identified with paperelectrophoresis and nine with cellulose acetate membraneelectrophoresis, the essential differences in the techniques and theapparatus employed being the result of the type and texture of thesupporting medium in those two instances.

Agar-gel electrophoresis provides a technique that is simple, rapid, hashigh resolving power and demonstrates the relative mobilities of themigrating proteins. With this technique, tissue fragments can be studiedelectrophoretically, and the agar-gel provides a sensitive technique forimmunoelectrophoresis. Five protein fractions can be identified fromnormal human serum with this technique.

In disc electrophoresis, advance preparation of a polyacrylamide gel inthree stages is necessary. The three layers, the top one of whichincludes the specimen, are placed in a tube and photopolymerized. Twentyprotein fractions of human serum can be identified with this method.

In starch-gel block electrophoresis, the migrating ions must passthrough the gel matrix, rather than in aqueous film around thesupporting material, as in the filter paper system. Starch-gel hindersthe migration of larger proteins more than smaller proteins. Exact andprecise preparation of the gel is essential and the nature of the starchitself is of primary importance. After electrophoresis, the

" gel is cut into thin layers and stained, then observed or scanned. Innormal human serum, 20 protein fractions can be identified with thismethod.

Starch block electrophoresis uses a homogeneous block of potato starch.The material to be fractionated is applied in a narrow slit and theblock is covered with a paraffin film to prevent evaporation. Afterelectrophoresis, the block is cut into short segments, the materialeluted, and analyzed. This technique is most useful for recoveringfractions of large molecular weight, which migrate freely in starchblock, and which are unable to pass through the mesh of gels. In normalhuman serum, five protein fractions can be identified with this method.

Sponge rubber electrophoresis provides a simple method of separatingprotein components of serum. The supporting medium does not complex withserum proteins and the sponge can be squeezed to obtain the proa. teinsafter separating. -Five protein fractions can be identified from normalhuman serum with this method.

In continuous flow electrophoresis, electric current is continuouslyapplied and the sample is continuously applied to the medium used,normally filter paper. The electromotive force is applied transverse tothe sample track, and as the sample travels in the medium, it spreadsfanwise. The study normally takes several days, and five proteinfractions of normal human serum can be identified.

SUMMARY OF THE INVENTION The present invention is directed to zoneelectrophoresis using a medium coating on a plastic film base. Itspecifically utilizes a thin layer of starch-gel medium adhered to anoptically transparent Mylar film base. This film base has been selectedfrom over 100 materials ranging from paper through glass and varioustypes of plastic, and has been found to have a superior ability toadhere the starch-gel medium in a desirably thin coating. Thisarrangement provides a test strip of starch-gel medium, which hasgreater sensitivity than paper or cellulose acetate media, while at thesame time afiording the convenience of automatic scanning of the teststrip for quantitative analysis of the sample material being tested.

As indicated above, know types of starch-gel media have been used inblock form, which is quite thick. Approximately 8 hours are requiredbetween the making of the gel and its use for testing. With the presentsystem, however, the starch-gel on Mylar strip can be used withinminutes from the time preparation is begun. Moreover, the knownstarch-gel blocks could not be scanned, whereas the present strips lendthemselves to the use of known photoelectric scanning techniques.

The Mylar base and starch-gel medium of this invention provide a matrixin which a density pattern of sample particles is developed from whichthe relative percentages of different fractions in a sample material,such as proteins that make up a serum, can be calculated. Moreover, withthis starch'gel and Mylar base medium, to 25 fractions can be separatedfrom normal serum, as compared with 5 fractions possible with apresently used paper media, of comparable convenience. Also, a pluralityof samples can be handled on a single sheet or film base.

In addition to the above, the present invention provides specificapparatus and techniques for facilitating the preparation, use, andscanning of a film supported medium. To this end, a drum or roller-typeapplicator is provided to assist in the coating of the Mylar base with athin film of starch-gel. A cutter board of novel construction isprovided for preparing the coated base and medium into usable strips forelectrophoresis. Also, a technique for glycerinating the starch-gel hasbeen developed to inhibit the tearing of the starch-gel film, maintainthe gel in adherence to the base film, and harden the gel so that thefinished strip can be run through an automatic scanner for analysis. Amat strip has been provided for use with the starch-gel film toaccommodate the conventional photoelectric scanners, which requirediffused light.

Basically, in the preferred mode of practicing this invention, a sheetof Mylar is coated with a thin film of starch-gel. The coated film isplaced on a cutting board and parallel channels are for-med to dividethe gel coating into strips. The gel coated film is then placed on asupport stand of an electrophoresis cell with one end of the filmconnected to a negative potential and another portion of the film spacedtherefrom connected to a positive potential. A sample is applied to eachstrip of the gel at a starting line, and a current is applied across thefilm. After a predetermined time during which the particles of thesample, such as serum proteins, migrate, the film is removed. The sheetis then stained, e.g., with protein stain. It is then destained to takeout the background color leaving the dye bound only to the differentfractions of the sample material in the medium. To facilitate handling,especially in automatic scanning equipment, the starch gel isglycerinated in accordance with a critical technique to be described indetail subsequently. The film is then cut into strips along the channelspreviously made in the gel, to facilitate scanning in an automaticphotocell analyzer. A matte strip is placed over the gel to diffuselightpassing through the optically clear Mylar film base, and thesandwich is fed through the analyzer. The dyed protein bands in the gelvary the amount of light passing through the strip. The lighttransmitted indicates the quantity and location of the various parts ofthe sample, which have migrated different distances during the time thatthe strip was in the electrophoresis cell.

Accordingly, it is an object of this invention to provide new andimproved methods and apparatus of electrophoresis that are convenient touse and sensitive in operation. Other objects and a more completeunderstanding of this invention will be obtained from the detaileddescription, when considered in connection with the accompanyingdrawings.

BRIEF DESCRIPTION OF THE DRAWINGS FIGURE 1 is a top plan view of anapplicator for applying starch-gel to a sheet of plastic film;

FIGURE 2 is a sectional view of the applicator of FIGURE 1, taken alongthe line 22 and looking in the direction of the arrows;

FIGURE 3 is a top plan view of a cutting board for preparing a coatedsheet of plastic film for use with the present system;

FIGURE 4 is a longitudinal sectional view of the cutting board of FIGURE3, taken along the line 44 and in looking in the direction of thearrows;

FIGURE 5 is a diagrammatic view of a pipette and vacuum source, used inthe preparation of a coated plastic film for use with the presentsystem;

FIGURE 6 is a diagrammatic perspective view of a sheet of plastic filmto be coated with a layer of starch-gel for use in the present system;

FIGURE 7 is a narrow plastic strip with a matte or diffusing surface foruse in the present system;

FIGURE 8 is a side elevational view, with parts removed and parts insection, diagrammatically illustrating a plastic sheet wrapped aroundthe applicator of FIG- URE 1;

FIGURE 9 is a diagrammatic view similar to FIG- URE 8, showing themanner in which a plastic sheet is removed from the applicator of FIGURE8 to coat the sheet with a layer of starch-gel;

FIGURE 10 is a diagrammatic perspective view of the cutting board ofFIGURE 3 and pipette of FIGURE 5, illustrating the manner in which thecoating on a sheet of plastic film is divided into spaced strips inpreparation for use;

FIGURE 11 is a diagrammatic perspective view of a coated sheet ofplastic film prepared in the manner illustrated in FIGURE 10;

FIGURE 12 is a transverse sectional view of the coated sheet of FIGURE11, taken along the line 12-12 and looking in the direction of thearrows;

FIGURE 13 is a diagrammatic perspective view of an electrophroesis cellfor applying an electric current to the starch-gel film of FIG-URE 11;and

FIG-URE 14 is a diagrammatic view, partly in Section, illustrating themanner in which a strip of the film in FIGURE 11 along with the mattestrip of FIGURE 7, are automatically scanned to determine densityvariations of dyed materials that have migrated within the layer ofstarch-gel adhered to the plastic film.

DESCRIPTION OF THE PREFERRED EMBODIMENT An applicator 20 is shown inFIGURE 1 for applying a coating of gel medium to a base sheet of plasticfilm. The applicator 20 consists of a drum 22 about which a plasticsheet will be wrapped for coating, a vessel 24 that holds a medium to beapplied to the plastic sheet and which also receives a portion of thedrum 22, and two end plates 26, 27 that support the drum and vessel.Preferably, the vessel is made of plastic that will withstandtemperatures of 200 to 210 degrees Fahrenheit at which starch-gel mediummay be when placed in the vessel. As best indicated in FIGURE 2, thevessel 24 is formed of a longitudinal segment of a cylindrical tube,oriented concave upward and extending between the two end plates 26, 27.The vessel is adhered to the two end plates and together with the endplates forms a support for the drum 22.

The drum 22 is cylindrical in shape, being in the form of a tube withcircular end walls 29, 30. A stud shaft 31 extends from the end wall 29,and a stud shaft 32 extends from the end Wall 30. These stud shafts arereceived in semi-circular recesses at the top of end plates 26, 27, onesuch recess being located at 34 in FIGURE 2. The drum 22 is supported bythe end plates 26, 27 so that the spacing between the drum 22 and vessel24 is somewhat greater at a central area 24c of the vessel than atlongitudinal edges 24a, 24b. In use, the drum is rotated in a directionfrom the vessel edge 24a to edge 24b and the greater spacing in thecentral area acts as a reservoir for the coating medium. The closespacing at 24b prevents spillage and helps control the coatingthickness.

A cutting board 38 is shown in FIGURE 3 for use in dividing the mediumcoated on a sheet of plastic film into spaced strips and later forcutting the film into strips. The cutting board 38 is preferably ofplastic and consists of a fiat rectangular panel 40 adapted to receive acoated sheet of plastic film in a flat condition. A raised border 42 isprovided about three sides of the fiat rectangular panel 40, andincludes three portions 42a, b, c. The panel has one open end at 40a.The surface of the panel 40, within the border 42, is constructed of asize to receive a sheet of plastic film material coated on theapplicator 20. A removable cross piece 44 extends across the panel 40and rests on the border portion 42a, 42c, and is spaced from the borderportion 42b and the open end 40a. Locating studs 45 on the borderportions 42a, 0, are received in apertures in the cross piece and locateit relative to the top border portion 42b. As shown in FIG- URE 3, thecross piece 44 and the border portion 42b are adapted to support aT-square 46. The T-square 46 will slide across the board 38 above thepanel 40, and is used to prepare lines in the coating of the plasticfilm that is placed on the panel 40.

A clamping member 48 is located at the open end 40a of the panel 40, andis used for cutting the coated film into separate strips afterelectrophoresis. The clamping member 48 includes a flat plate 49 thatextends across the panel surface 40, and fits closely within the borderportions 4211, 420. The fiat plate 49 is relatively narrow and isthinner than the thickness of the raised borders 32a, 42c so that it canbe supported flush with the raised borders and yet provide space betweenit and the upper surface of the panel 40 to receive a sheet of plasticfilm material coated with a medium, such as a starch-gel. A rod 50 ofcircular cross section extends longitudinally d Wn the middle of thefiat plate 49 and across the panel 40. Opposite ends of the rod 50 restupon raised border portions 42a, 42a. The rod 50 is secured to the uppersurface of the flat plate 49 and thereby supports the plate above thepanel 40. Spaced pairs of blocks 51, 52 on the border portions 42a, 420,respectively, locate the extending ends of the rod 50 to maintain theclamping member 48 in proper location, but pivotable about the axis ofthe supporting rod 50. When the clamping member 48 is pivoted about theaxis of the rod 50, it presses against the film on the panel 40, tosecurely hold the film during a cutting operation. It will then pivot toa horizontal position, to allow the film to be moved relative to thepanel 40.

A channel former 56 is shown in FIGURE 5, and is used to make channelsin the medium coated on the sheet of plastic film used in the presentsystem. The channel former 56 is preferably a glass capillary pipette 58connected by a vacuum hose 59 to a vacuum source, such as a vacuum pump60. The pipette 58 has a small open end 6.1 approximately 1 millimeterin diameter that can be guided along the T-square 46 of the cuttingboard 38 to form channels in a medium on a plastic film, as will beexplained in more detail subsequently in connection with the method andoperation of the present system.

A sheet of film 66 is shown in FIGURE 6 of the drawings, and isconstructed to be coated with a medium in which particles can migrateunder the influence of an electrical field. When coated, it is used toseparate particles by electrophoresis.

The film 66 is formed of two separate pieces, a large piece 67 to becoated and small manipulator piece 68. The large and small pieces are ofequal width, and abut each other as illustrated. A strip of tape, forexample, cellophane adhesive tape 69 extends across the upper surface inthe orientation of FIGURE 6, and adheres the two pieces 67, 68 together.The large piece 67 when coated is used to provide several narrow stripsfor re ceiving samples of materials to be analyzed. The smaller piece 68is used as a leader to facilitate coating the larger piece on theapplicator 20 of FIGURES 1 and 2. The width of the supporting film 66corresponds essentially to the width of the panel 40 within the raisedborder portions 42. A width suitable for making a plurality of smallerstrips is preferred, for example a width of 11 /2 inches is particularlysuitable for providing five spaced test strips and can be received onexisting equipment for applying a current across the gel. Suitably, thelength of the large piece 67 can be 14 /2 inches and the length of thesecond smaller piece can be 5 inches.

For optimum results in separating ions of diiferent mobility, inaccordance with this invention, a starch-gel medium is used. With thispreferred medium, only a supporting film 67 made of polyethyleneterephthalate, such as Mylar, has been found to be completely suitable.The discovery of the suitability of this film material, its use tosupport a starch-gel medium, and its superiority over other possiblesubstrates for supporting a layer of starch-gel for electrophoresisconstitutes an important aspect of this invention. This Mylar filmmaterial is optically clear, strong, and tenaciously adheres a medium ofstarch-gel especially suitable for the present pr cess. Preferably, asheet of film 0.005 inch thick is used.

A narrow plastic strip 70 is shown in FIGURE 7 for use with test stripsprepared from the large piece 67 of the supporting film 66 when thelarge piece is cut into separate test strips and analyzed afterelectrophoresis. The strip 70 preferably has one surface 71 of a mattefinish, or the strip is otherwise translucent. When this strip is placedover the medium on the optically clear supporting film 67, it diffuseslight and facilitates scanning by photoelectric detectors that areconstructed to operate with diffused light. Preferably, the strip 70 isof polyethylene terephthalate, such as Mylar.

The manner in which the above-described apparatus is used and the methodof practicing the present invention is best understood in connectionwith FIGURES 8 to 14 of the drawings.

The drum 22 is removed from the applicator 20 and the compositesupporting film 66 is fastened to the drum, as by a piece of cellophaneadhesive tape. The supporting film is attached at one end of the largepiece 67 and is then rolled about the drum, so that the smallmanipulator piece 68 forms a partial cover over the large piece 67 onthe drum. The outer end of the manipulator piece 68 is then adhered tothe rolled film, as by a small piece of adhesive tape to hold the filmwrapped about the drum.

A starch-gel medium is prepared and placed in the vessel 24. The mediumis shown in the vessel 24 in FIG- URE 8, and indicated by the referencenumeral 75. The drum 22 with the rolled supporting film 66 is thenplaced within the vessel 24, supported on the two end plates 26, 27 soas to be spaced from the vessel 24 but partially immersed in thestarch-gel 75. When the drum 22 is placed into position, the terminalend of the film 66 (i.e., the outer end of the manipulator piece 68) islocated out of the gel, and pointing upward, as illustrated in FIG- URE8. The outer end is then loosened from the drum 22, and pulled aroundand over the drum, as illustrated in FIGURE 9. As a result of thismanipulation, the surface of the film 66 facing the drum 22 remains freeof starch-gel, while the outer surface is coated with a continuous layer76 of starch-gel of essentially uniform thickness preferably 1millimeter thick. The manipulator piece 68 serves to initially removeany air bubbles in the starch-gel so the coating on the piece 67 isuniform.

By way of a specific example, the starch-gel 75 with which the sheet orfilm 66 is coated is prepared by diluting 15 ml. concentrated TrisBuffer (No. 301), manufactured by The Spel Systems, Cleveland, Ohio,with 135 ml. of distilled water, and mixing the diluted butter with 18grams Connaught Hydrolyzed Starch Powder in a two liter round bottomfiask so that the starch is evenly distributed. The starch is heated tothe boiling point with constant swirling. The boiling is continued for afew seconds only, and the flask is then connected to a water pump toremove all air bubbles. This entire procedure takes less than 2 minutes,consisting of 70 seconds heating plus 20 seconds degassing. The hot gelat about 207 degrees Fahrenheit is then poured into the vessel 24 at aneven rate to avoid entrapping air bubbles. The drum 22 wrapped with thefilm 66 is then placed into position in the vessel so that the outer endof the film is facing up ward about /2 inch from the gel surface. Thetape adher ing the outer end of the film to the drum is removed and thefilm is rolled off the drum with constant speed and pulled onto ahorizontal, smooth, surface. The upper surface only of the film iscoated with the gel to a thickness of 1 millimeter. After one minute,the tape 69 connecting the small piece 68 to the large piece 67 isremoved and the small piece of film is discarded. The gel on the film isnow ready to be divided into spaced strips.

To prepare the layer of gel 76 on the film 67, portions of the gel areremoved to form channels 78 in a pattern that is best shown in FIGURE 11where spaced test strips 79a, b, c, d, e are shown separated by narrowerstrips 80, all defined by the channel '78. As shown, the channels 78 donot extend the full length of the film 67. In practice the length of thechannel 78 depends upon the length of strip 79 desired, which in turnWill depend upon the size and construction of the electrophoresis cellor apparatus used to apply a current to the gel medium on the film.

The manner in which the channels 78 are formed is shown in FIGURE of thedrawings. The supporting film 67 with the layer of gel 76 is placed onthe panel 40 of the cutting board 38. The film is located within theraised border 42 on three sides of the cutting board, and extendsslightly from the open end 40a. The cross piece 44 is then placed acrossthe border portions 42a, 420. The T- square 46 is positioned to extendalong the cutting board 40, resting on the raised border 42b and thecross piece 44. If desired, the flat rectangular panel 40 can be linedor a pattern can be placed beneath it to provide a guide for determiningthe location of the channels 80 to be formed. Channels 80 are formed byplacing the T-square 46 at a desired location, and removing the gel 76along a line defined by the T-square parallel to the side borderportions 4211, 420. This is best accomplished with the capillary pipette58, shown in FIGURE 5. The open end 61 is placed against the gel surface76 at the top of the cutting board 38, just inside the border portion42b. The pipette is held at an angle of about 20 degrees from thehorizontal, pointing in the direction in which it is to be moved alongthe film 67. The vacuum pump 60 is energized, and the pipette is movedalong the film, being guided against the straight edge surface of theT-square 46.

The entire preparation of the starch-gel film is accomplished whileavoiding drying the gel, and takes about 10 minutes. As illustrated inFIGURE 12, the channel 78 extends completely through the gel layer 76 soas to isolate the strips 79 from the gel adjacent thereto. This preventsseparate samples from spreading to adjacent strips.

After the layer of gel has been channelled into spaced strips ofstarch-gel on the film 67, the coated film is placed 8 in anelectrophoresis cell 85, shown in FIGURE 13. The electrophoresis cellsupports the film 67 draped over a horizontal support rod 86, so thatopposite ends of the film 67 extend down into a reservoir of electrolytein a base 87 of the electrophoresis cell. Two electrodes are provided inthe cell, one at one end of the strips 79 and the other at the other endof the formed strips, in the container 87. A wick (not shown) ismaintained in contact with the gel at each end of the film 67 to holdthe film firmly in position and connect the film electrically withelectrodes.

A material to be analyzed, such as serum proteins, is applied to each ofthe spaced test strips 79ae at the top of the electrophoresis cell 85. Astarting line may be marked for reference. An electrical current is thenapplied along the test strips 79 to cause ionized particles of testmaterial to migrate through the starch-gel medium along the the teststrips 79a-e. Different particles, due to their different size andmobility in the starch-gel medium, migrate different distances in anyallotted time and thereby become separated from other particles ofdifferent types.

By way of a specific example, five samples of ten lambda serum areplaced at the top of the test strips 79ae adjacent a starting line,which can be applied with a chinamarking pencil. The serum is appliedcarefully so that the gel is not cut during the application. A spot ofreference dye is placed at the starting line, on one edge of the gel, toact as an indication of how far fractions of the test material aremigrating. With a Tris-Borate Buffer system by way of example, the serumproteins will migrate from the negative to the positive pole when adirect electric current is applied along the test strips. The positivelead of a power supply is therefore connected to the lower side of thefilm at the far ends of the channels and the negative lead of the powersupply is connected at the end of the film 67, where the channels werenot completed, or alternatively, at the starting line on the strips ifthe electrophoresis cell is constructed to facilitate this. With thepresent example, only those portions of the strips 79 are being usedthat extend from the top of the cell 85 to the lower end of the film 67,where the channels 78 extend to the end of the film. With the cellmaintained at room temperature, 68 degrees Fahrenheit, a direct currentof volts and 4 milliamps are applied to the electrophoresis cell.

The current is applied for approximately 16 to 17 hours, and at the endof this time the reference dye migrates 10 to 11 centimeters from theapplication point. During this time, different proteins of the serumwill migrate to different extents, depending upon their characteristics.

The starch-gel film 67 is then removed from the electrophoresis cell 85and placed on the cutting board 38, which has been moistened with waterto hold the film in position. The film is placed with the starch-gelside up and with the portion that has not been channelled into stripsextendlng from the open end 40a of the board. The unchannelled portionof the film is cut away.

The starch-gel film is then placed in a protein staining solution tostain the proteins. The starch-gel sheet is then d e-stained to clearthe background, using standard technrques.

By way of specific example, the starch-gel film is immersed in aconventional protein stain for approximately /2 hour. It is thende-stained as follows: A stock solution of methyl alcohol, distilledwater and glacial acetic acid in proportions by volume of 5 :5 :1 isprepared. A de staining solution is made by mixing 600 ml. stocksolution plus 400 ml. of distilled water. The starch-gel sheet istransferred from the staining solution to the de-staining solution for aperiod of 2 to 3 hours. After de-staining, only the dye bound to thedifferent fractions of the test material remains.

To further facilitate handling of the starch-gel sheet in the presentsystem, the gel is glycerinated. This step is important to assure thatthe starch-gel can be handled without damaging the test strips. By wayof specific example, the starch-gel sheet is placed in an 8.0% glycerinesolution (80% glycerine, 20% water, by weight) heated to 80 degreescentigrade. The starch-gel sheet remains in the solution for one (1)minute, the excess glycerine is then wiped from the sheet and the sheetis left at room temperature for /2 hour. The temperature at which theglycerine is maintained is critical. The temperature must be 80 degreescentigrade, although a variation of not more than :1 degree centigrademay be acceptable. At lower temperatures than 80 degrees centigrade, theglycerine will not penetrate evenly into the gel, even for times of onehour or more. If the temperature is higher than 80 degrees centigrade,air bubbles in the gel explode, or otherwise damage the gelunacceptably. When the glycerinated starch-gel sheet is removed, itbecomes plasticized, rendering it resistant, hardened, and betteradhered to the film base. As a result, it can be handled and scanned inautomatic scanning equipment.

Scanning is accomplished in a conventional recording scanner andintegrator that utilizes a light source and a photocell. One suitableanalyzer is a model R Spinco Analytrol, sold by Beckman Instruments,Inc., Palo Alto, Calif.

To prepare the starch-gel sheet for analysis, it is again placed on thecutting board 38, this time with the channels 78 parallel with the openend 40a. The channels are aligned with the end 40a of the panel 40,clamped in place by the flat plate 49 of the clamping member 48, and thefilm is cut along the channels, using the flat plate 49 as a guide. Eachcut strip 79 is then separately analyzed by photoelectrically scanningit in an analyzer. This is accomplished as diagrammatically shown inFIGURE 14. The matte strip 70 is placed over the starch-gel strip 79a,with the starch-gel sandwiched between the supporting film 67 and thematte strip 70. The sandwich strip is moved by rolls 90 between a lightsource 91 and photocell 92 connected with an amplifier 93 that controlsa recording pen (not shown), which automatically records on a chart atrace of color density verses distance along the test strip 79a. Thearea under the curve is then integrated to facilitate measuringconcentration of each component of the test material.

In summary, a new system of electrophoresis is provided, using improvedapparatus and techniques that facilitate the preparation and use ofimproved test strips. Thus, it will be apparent that an important aspectof the present system is the provision of a test strip comprised of astarch-gel medium adhered to a film of polyethylene terephthalate,thereby providing a convenient strip with a medium for separatingmaterials to be analyzed into component parts. Such a starch-gel stripcan separate 15 to 25 fractions of a protein, whereas paper stripshaving similar convenience in handling and use, will separate only 5fractions. As compared with a starch-gel block, a sensitive medium isprovided in a new manner which permits the preparation test strips, anduse within approximately minutes of the starting time, whereasstarch-gel in block form requires at least 8 hours between the making ofthe gel and the using of it in actual tests. Moreover, in strip form asprovided with this invention, the separation of materials can be scannedwith an automatic photocell scanner to provide a pattern of densitydistribution. From this, a relative percent of different fractions ofproteins for example can be easily calculated. The preparation and useof such test strips is facilitated by the apparatus and methodsdescribed in detail for coating a sheet of plastic film material,providing channels in the sheet, glycerinating the starch-gel tofacilitate handling the test strip, and providing a matte strip forfacilitating the use of the optically clear base film in scannersconstructed for use with diffused light.

While a preferred embodiment of this invention has been described indetail, it will be apparent that various modifications or alterationsmay be made therein without departing from the spirit and scope of theinvention, as set forth in the appended claims.

What is claimed is:

1. In a method of preparing an article for separation analysis ofmaterials, especially biochemical materials, by electrophoresis, thesteps comprising:

(a) connecting one end of a sheet of plastic film to a horizontalcylindrical drum,

(b) winding the sheet about the drum,

(c) dipping a portion of the drum across the entire width into astarch-gel medium useful for separation analysis of materials byelectrophoresis, with the outer end of the sheet out of the material,

(d) pulling the outer end of the sheet and rolling the drum and sheetthrough the medium at a substantially constant speed so that the sheetis unwound from the drum and becomes spaced from the drum only above themedium,

(e) whereby the outside surface of the sheet of plastic film comes incontact with the medium while the inside surface does not and the sheetof film material is uniformly coated on one surface only with themedium.

2. The method of claim 1 wherein the sheet is comprised of two separateparts temporarily fastened together, a first part attached at one end tothe drum and a second manipulating part removably attached to a free endof the first part and wherein the second part is drawn through thestarch-gel medium prior to the first part to remove bubbles that mayexist in the medium and is thereafter removed from the first part.

3. The method of claim 1 wherein the starch-gel medium is at atemperature of between 200 and 210 degrees Fahrenheit.

4. The method of claim 1 including the steps of:

(f) forming parallel channels in the starch-gel along a portion of thefilm to provide spaced strip portions of starch-gel on a single sheet offilm,

(g) depositing a material to be analyzed upon the strip portions of thestarch-gel,

(h) subjecting the strips to an electric field until particles of thematerial being analyzed migrate along the strips,

(i) coloring with a stain the material being analyzed in the starch-gel,

(j) glycerinating the starch-gel,

(k) cutting the film into separate strips along the channels, and

(l) scanning a strip photoelectrically to determine the variations incolor density along its length.

5. The method of claim 1 including the steps of forming parallelchannels in the gel medium along a portion of the film to provide spacedstrip portions of gel medium on a single sheet of film.

6. The method of claim 5 wherein the parallel channels are formed bymoving a pipette through the layer of gel medium and drawing awayportions of the layer with a vacuum.

7. The method of claim 5 including the steps of:

(c) depositing a material to be analyzed upon the strip portions of thegel medium,

(d) subjecting the strips to an electric field until particles of thematerial being analyzed migrate along the strips,

(e) coloring with a stain the material being analyzed in the gel medium,

(f) glycerinating the gel medium,

'(g) cutting the film into separate strips along the channels, and

(h) scanning a strip photoelectrically to determine the variations incolor density along its length.

8. The method of claim 1 including the steps of providing glycerine at atemperature of degrees centigrade placing a starch-gel coated sheet inthe glycerine for 1 1 12 approximately one minute and then removing thesheet 3,061,472 10/1962 Brockway 117139.5 and allowing it to dry.3,303,044 2/ 1967 Fenley 11734 9. The method of claim 8 wherein theglycerine is a solution of 80% glycerine and 20% water, by volume. JOHNMACK, Primary E i References Cited 5 A. C. PRESCOTT, Assistant ExaminerUNITED STATES PATENTS US. Cl. XR 343,375 6/1886 Howe 117-115 117 115 13;1 5 2,843,540 7/1958 Ressler 204-180 2,962,425 11/1960 Sharpsteen eta1. 204180 10 UNITED STATES PATENT OFFICE (s/ss CERTIFICATE OF C R NPatent No. 3, -97, l- &l Dated Februa 21;, 1970 nvem fl I JOSeph PaksiIt is certified that error appears in the above-identified patent andthat said Letters Patent are hereby corrected as shown below:

Column 5, line th, "32a" should be 42a Column 7, line 37, "channel"should be channels Column 7, lines 70-71, "channel 78 extends" should bechannels 78 extend Column 10, line 51, "steps should be step A 2: 2;.El-3 :17; 1 '0 n w k L if? v SIGNED AND $EME Amt:

Edward 1!. mad-er, Ir-

Mmmllg 0mm IRMA! I. samrmm, m.

' dominion of Patents

